Int. J. Oral Maxillofac. Surg. 2014; 43: 514–521 http://dx.doi.org/10.1016/j.ijom.2013.10.017, available online at http://www.sciencedirect.com
Research Paper Bone Substitutes
Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells
A. Konermann1, M. Staubwasser2, C. Dirk3, L. Keilig3, C. Bourauel3, W. Go¨tz1, A. Ja¨ger1, C. Reichert1 1
Department of Orthodontics, Dental School, University of Bonn, Bonn, Germany; 2Institute of Geology and Mineralogy, University of Cologne, Cologne, Germany; 3Oral Technology, Dental School, University of Bonn, Bonn, Germany
A. Konermann, M. Staubwasser, C. Dirk, L. Keilig, C. Bourauel, W. Go¨tz, A. Ja¨ger, C. Reichert: Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells. Int. J. Oral Maxillofac. Surg. 2014; 43: 514–521. # 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The aim of this study was to determine the material composition and cellmediated remodelling of different calcium phosphate-based bone substitutes. Osteoclasts were cultivated on bone substitutes (Cerabone, Maxresorb, and NanoBone) for up to 5 days. Bafilomycin A1 addition served as the control. To determine cellular activity, the supernatant content of calcium and phosphate was measured by inductively coupled plasma optical emission spectrometry. Cells were visualized on the materials by scanning electron microscopy. Material composition and surface characteristics were assessed by energy-dispersive X-ray spectroscopy. Osteoclast-induced calcium and phosphate release was material-specific. Maxresorb exhibited the highest ion release to the medium (P = 0.034; calcium 40.25 mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (P = 0.021; calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (P = 0.034; calcium 16.34 mg/l day 5, phosphate 30.6 mg/l day 5). All investigated materials showed unique resorption behaviours. The presented methodology provides a new perspective on the investigation of bone substitute biodegradation, maintaining the material-specific micro- and macrostructure.
Bone is a dynamic tissue remodelled by osteoblast–osteoclast interactions under the regulation of the RANK/RANKL/ OPG system.1 In bony defect healing, bone grafting materials are the most important acquirement in osseous tissue regeneration. The osteoconductive and 0901-5027/040514 + 08 $36.00/0
osteoinductive features of these materials are controlled by different inherent material characteristics as much as by environmental criteria.2 The most important requirements for an ideal bone substitute are biocompatibility, osteoinduction, and osteoconduction, and for dental implants,
Key words: bone substitutes; biodegradation; calcium; osteoclastic cells; phosphate. Accepted for publication 17 October 2013 Available online 20 November 2013
stable long-term osseointegration with the re-establishment of structural tissue to attain a ‘restitutio ad integrum’.3 Previous in vitro research has mainly focussed on osteoblast–material interactions,4–6 and investigations of bone substitute biodegradation have principally
# 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Bone substitute resorption been restricted to animal experiments.7,8 Osteoclastic bone resorption occurs following strong attachment of the osteoclast to the hard tissue surface and subsequent generation of an acidic pH of 4.5 within the ‘ruffled border’, a special cellular compartment, to solubilize the alkaline salts of the bone mineral.9 Prior investigations of bone graft–osteoclast interactions have tended to study the cellular characteristics of bone remodelling processes,10–12 but the physico-chemical dissolution behaviour of bone substitutes has yet to be fully determined. Bone substitute materials include both synthetic and biological substitutes, and are characterized by their chemical composition, crystallinity, and morphology13; these have to meet specific criteria, as defined by their surface structure and configuration, in order to mimic in vivo effects.14 Calcium (Ca)–phosphate (P) bioceramics of synthetic or natural origin have unique compositions analogous to bone mineral and its specific turnover, which involves, among other processes, physiological Ca and P metabolism.14 However, the specific mechanisms that underlie these remodelling processes have yet to be fully elucidated. The aim of this study was to quantify the cell-mediated dissolution behaviour of three different bone substitutes in vitro by osteoclasts, with regard to Ca and P release from the bone substitutes. Furthermore, the cell adhesion qualities and the material composition of the bone substitutes were investigated. Materials and methods
Cell culture
A RAW 264.7 murine monocytic/macrophagic-type cell line (CLS Cell Line Services GmbH, Eppelheim, Germany) was seeded at a density of 3 107 cells/ml and cultured with 20 ng/ml macrophage colony-stimulating factor (M-CSF; BioCat GmbH, Heidelberg, Germany) and 30 ng/ml RANKL (Axxora GmbH, Lo¨rrach, Germany) in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated foetal calf serum (FCS; Invitrogen) and 1 mg/ml penicillin/streptomycin (Invitrogen) at 37 8C in a humidified 5% CO2 atmosphere for osteoclast differentiation, as described previously.19,20 After 12 days, tartrateresistant acid phosphatase (TRAP) staining, as well as measurement of osteoclastic marker expression (TRAP, cathepsin K, vitronectin receptor) was performed to determine the osteoclastic phenotype (data not shown). The cells were then passaged into 24-well tissue culture plates (Greiner Bio-One International AG, Kremsmu¨nster, Austria), covered with or without different Ca–P-based bone substitutes in 2 ml medium/well. All materials were obtained directly from the manufacturers in sealed vials and were used without further modification. Control assays were performed with the addition of 100 nM bafilomycin A1 (Sigma–Aldrich, St. Louis, MO, USA) to wells containing osteoclasts, in order to mediate inhibition of active endocytosis by RAW cells (data not shown).21,22 Samples with medium alone served as reference controls.
Bone substitutes
Three different bone substitutes were included in this study, one of bovine origin and two synthetic composite materials: Cerabone1 (Botiss Dental GmbH, Berlin, Germany), a high-temperature sintered material of bovine origin enclosing highly crystalline hydroxyapatite (HA) as the sintered inorganic part of bovine bone, with 50% porosity and particle sizes of up to 1 mm15; Maxresorb1 (Botiss Dental GmbH, Berlin, Germany), a synthetic material composed of 60% HA/40% btricalcium phosphate (b-TCP) with macropores of 200–800 mm and micropores of 1–10 mm16; NanoBone1 (Artoss GmbH, Rostock, Germany), a bone substitute based on nanocrystalline HA embedded in a nanostructured silica, which has been described to undergo complete biodegradation by osteoclasts in vivo.7,8,17,18
Sample preparation
After 1 and 5 days, cell culture supernatants were harvested and transferred into glass vials for further treatment. Samples were dried using a red light (150 W) under a laboratory fume hood; 0.2 ml of aqua regia was then added (mixed from 3 parts 65% hydrochloric acid and 1 part nitric acid; Merck KGaA, Darmstadt, Germany) and the samples left for 24 h. This was followed by a final dilution in 3 ml Ampuwa water (Fresenius Kabi Germany GmbH, Bad Homburg, Germany) to filter out any residue with a particle diameter over 1.2 mm remaining after sample drying (FP 30 1.2CA; Schleicher & Schuell BioScience GmbH, Dassel, Germany) in glass containers (bottles with a rolled edge, 3 ml; VWR International GmbH, Darmstadt, Germany).
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Inductively coupled plasma optical emission spectrometry analyses
Ca and P ion concentrations in the samples (dilution 1:10) were measured with an inductively coupled plasma optical emission spectrometer (Ametek, Spectro A. I. Inc., Mahwah, NJ, USA) and an SC-4 DXS autosampler (Elemental Scientific, Omaha, NE, USA) at wavelengths of 317.933 nm (Ca) and 177.495 nm (P). The control program of the mass spectrometer performed three consecutive measurements of each sample, delivering the mean and standard deviation of the three results. Ion concentrations were calculated by inductively coupled plasma optical emission spectrometry (ICP-OS), which was calibrated by a standard control dilution series of defined ion concentrations subjected to matrix adjustment, delivering calibration curves as a reference. The detection limit and high resolution were guaranteed by continuous unit calibration before measurement of the first sample and after every 10 samples, and by repeated measurements of random samples as quality control. Scanning electron microscopy analyses
After removal of the supernatants for ICPOS analyses, the cells cultivated on bone substitutes for 5 days, as well as the surfaces of the three bone substitutes, were visualized with a scanning electron microscope (SEM, XL30; Philips, Eindhoven, the Netherlands) operating at 10 kV. For this purpose, the cells were fixed with paraformaldehyde (2.5%, pH 7) at 4 8C for 24 h and dehydrated in a graded alcohol series for 1 h each (30%, 50%, 70%, 80%, 90%, and 99.6%). The samples were then immersed in 1,1,1,3,3,3-hexamethyldisilizane (HDMS; Carl Roth GmbH, Karlsruhe, Germany) for 5 min, air dried at room temperature, and mounted on stainless steel stubs with double sticky tabs. The conduction was increased with Leit-C (Conductive Carbon Cement; Plano, Wetzlar, Germany) and sputter coating with platinum (Sputter Coater S150B; Edwards Ltd, Crawley, UK). Energy-dispersive X-ray spectroscopy analyses
To assess the material composition of the three different bone substitutes, energydispersive X-ray spectroscopy (EDX) analyses were performed using a GENESIS 4000 EDX system (EDAX, Mahwah,
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NJ, USA) attached to the scanning electron microscope described above. For the EDX scans, an acceleration voltage of 20 kV and a magnification of 1000 were used in the SEM. The scan was performed over the whole visible region of the sample, thus determining the average element distribution in the region studied.
Table 1. ICP-OS ion concentration measurements.a Ion Ca
Results ICP-OS analyses
The absolute values of the ICP-OS ion concentration measurements are listed in Table 1. Measurement of Ca and P ion concentrations in samples containing bone substitutes in medium alone showed a slight initial decrease in the basal ion concentrations induced by Cerabone, NanoBone, and Maxresorb after 1 day of incubation. Ca concentrations declined from 27 mg/l as the standard medium control value to 29% thereof for NanoBone, 64% for Maxresorb, and 75% for Cerabone. Similarly, P concentrations diminished from 43 mg/l as the pure medium control value to 23% thereof for NanoBone, 79% for Cerabone, and 88% for Maxresorb. At day 5, these levels remained relatively stable for both Ca and P. Taken together, Maxresorb showed the highest Ca and P medium content, followed by Cerabone and NanoBone. Analyses of the influence of osteoclasts on the dissolution behaviour of bone substitutes showed an initial Ca and P decrease in the medium on day 1, as seen for bone substitutes in medium alone. In combination with Cerabone, the presence of osteoclasts led to lower ion levels compared to the bone substitute alone, whereas incubation of osteoclasts with NanoBone resulted in higher Ca and P levels. For Maxresorb, ion concentrations were lower in the presence of cells on day 1, but had become higher on day 5 compared to the bone substitute incubated alone. In detail, Cerabone and NanoBone exhibited similar concentrations developing for both Ca and P and a subsequent
BS
BS + OC + bafilomycin
P
Concentration (mg/l)
Material
BS + OC
Statistical analysis
Statistical tests were performed using GraphPad Prism software version 4 (MacKiev Software). Data are expressed as the mean standard deviation. Statistical significance was assessed by Student’s t-test for unpaired observations after verifying normal distribution of the values. P < 0.05 was considered statistically significant.
Condition
BS BS + OC BS + OC + bafilomycin
Day 1
Day 5
Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone
20.12 17.28 7.71 18.13 18.90 7.81 16.42 15.03 9.08
16.60 17.03 6.46 16.34 40.25 8.43 13.63 29.46 12.06
Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone
34.11 37.98 9.82 29.26 39.78 11.02 26.74 26.10 11.12
31.10 55.18 12.77 30.60 102.08 15.15 26.09 66.42 17.21
ICP-OS, inductively coupled plasma optical emission spectrometry; BS, bone substitute; OC, osteoclasts. a Absolute values of the ICP-OS ion concentration measurements are presented in mg/l. Data represent the mean values of three independent measurements for each sample.
Table 2. P-values for the analysis of the ICP-OS measurements: comparison between Cerabone, NanoBone, and Maxresorb for the measured Ca and P ion concentrations on day 1 and day 5.
Cerabone Cerabone Maxresorb
P
Ca
Material Maxresorb NanoBone NanoBone
Day 1
Day 5
Day 1
Day 5
0.773 0.021* 0.021*
*
*
0.034* 0.021* 0.034*
0.034 0.021* 0.034*
0.021 0.021* 0.021*
ICP-OS, inductively coupled plasma optical emission spectrometry. * P < 0.05.
Table 3. P-values for the analysis of the ICP-OS measurements: comparison between day 1 and day 5 for the measured Ca and P ion concentrations with Cerabone, NanoBone, and Maxresorb. Material Cerabone Maxresorb NanoBone
Ca (day 1–day 5)
P (day 1–day 5)
0.248 0.034* 0.773
0.386 0.034* 0.149
ICP-OS, inductively coupled plasma optical emission spectrometry. * P < 0.05.
decline to the initial levels on day 5. Maxresorb manifested a different dissolution pattern, as Ca increased to 149% and P to 238% of the medium control value on day 5. Statistical analysis revealed significant differences in both Ca and P content in the supernatants between the materials investigated and at both observation timepoints, except Cerabone in comparison to Maxresorb for Ca on day 1. Furthermore, Maxresorb showed statistically
significant differences in the measured values of both ions when compared between day 1 and day 5. Calculated Pvalues are listed in Tables 2 and 3. Data on the outcomes when bafilomycin A1 was added, showed that Ca and P release were suppressed by the inhibition of cellular activity due to exposure to the substrate. Only cells incubated with NanoBone could not be blocked effectively by bafilomycin, which became particularly evident on day 5 (Fig. 1).
Bone substitute resorption
Figure 1. Ca and P levels induced by bone substitutes and by bone substitutes cultured with osteoclasts. Results of the inductively coupled plasma optical emission spectrometry (ICP-OS) measurements of the Ca (a) and P (b) ion concentrations in samples containing DMEM with Cerabone, NanoBone, or Maxresorb, with or without osteoclasts, after 5 days of incubation. Measurements were taken at wavelengths of 317.933 nm for Ca and 177.495 nm for P. Concentrations are expressed in mg/l and represent the mean values of three independent measurements.
SEM analyses
EDX analyses
SEM analyses highlighted the different ultrastructures of the three bone substitutes investigated. Cerabone presented a compact but ruffled surface, with round protrusions observed at the highest magnification. In contrast to NanoBone and Maxresorb, this material had only a few pores. In the overview, Maxresorb was found to be widely covered with pores and NanoBone was covered with pores of different sizes. The highest magnification revealed that both materials were covered with micropores over the whole surface. Cellular adhesion to the surface was evident for all the materials investigated, and the cells appeared to be vital (Fig. 2).
EDX analyses confirmed Ca and P as the main structural substrates of the three bone substitutes evaluated (Fig. 3). In addition, NanoBone was shown to incorporate appreciable amounts of carbon (C) and silicate (Si). The perceptual net weights of the different materials were: Cerabone C 9.44, N 2.71, O 29.97, Na 0.24, Si 0.61, P 18.25, Ca 38.73; Maxresorb C 7.95, N 4.98, O27.5, Na 0.71, Si 0.23, P 17.93, Ca 40.69; NanoBone C13.2, N 3.81, O 32.61, Na 0, Si 10.01, P 11.62, Ca 28.75. Discussion
Bone grafting materials are the most important acquirement in osseous tissue
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regeneration, with autogenous bone still representing the gold standard due to its osteogenic, osteoinductive, and osteoconductive qualities and high biocompatibility.15 As its availability is limited, bone substitutes with identical or at least analogous capacities are useful alternatives to autogenous bone.13,15 The aim of this study was to elucidate the biochemical properties of three different Ca–P-based bone substitutes with regard to material characteristics, Ca and P metabolism, and interaction with osteoclasts. Ca–P bioceramics are subject to two basic degradation mechanisms, namely hydrolysis of Ca and P by extracellular fluids and cell-mediated degradation by macrophages, polymorphonuclear giant cells, and osteoclasts.23 The degraded products are resorbed and transcytosed to the extracellular space,9,24 which enables the concentration changes in Ca and P in culture supernatants to be assessed as an indicator of osteoclastic activity. In our experiments, an initial decrease in Ca and P content in the supernatants both with and without osteoclasts was evident and remained relatively stable over time. Maxresorb exhibited the highest ion content in the medium (calcium 40.25 mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (calcium 16.34 mg/l day 5, phosphate 30.60 mg/l day 5). In vivo, bone and bone substitute degradation is regulated by both cellular function and the interaction of a plurality of proteins, e.g. osteopontin, secreted by macrophages,25,26 whose cofactorial effects are difficult to replicate in an in vitro approach. However, our experimental setup maintained the specific features of the commercially available bone substitutes in accordance with their in vivo application. Other study designs have been based on morphological or morphometric analyses of osteoclasts or resorption pits on flat ceramic surfaces, thus discarding the specific structure of the bone substitute.27 All the materials investigated in this study show a specific micro- and macrostructure, supposedly exerting different effects on the dissolution behaviour of Ca and P. These ions, which have been proved to be a prerequisite for the process of osteoinduction, were determined to be the main structural substrates of the three bone substitutes evaluated, and NanoBone, in addition, contained considerable fractions of
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Figure 2. Ultrastructure and cell affinity of Cerabone, NanoBone, and Maxresorb. Representative visualization of RAW 264.7 cells on the bone substitute after 5 days. As well as the cellular morphology, the macrostructure of the different materials is evident. (a) Cerabone presented a compact, but ruffled surface. The cells were widespread and had thin protrusions. Several accumulations of cells located in resorption pits were visible. (b) In the ultrastructure, Maxresorb was covered with small pores. The cellular adhesion on Maxresorb was rather spare. Multinuclear cells were evident in large resorption pits. (c) NanoBone was ruffled and the material showed an inhomogeneous structure. It was completely penetrated by sponge-like holes. The cells on the material showed a strong polarization, and strong attachment to the surface was evident.
C and Si. Ca and P release by bone substitutes after implantation is followed by precipitation of an apatite layer due to supersaturation of these ions in the concavities of the material, presumably inducing osteoblastic cell formation directly or indirectly via the activation of growth factors.28,29 Our SEM analyses showed Cerabone to possess a ruffled surface with round protrusions and few pores, whereas Maxresorb was widely covered with pores and NanoBone was covered with pores of different sizes. These specific morphologies appear to determine the suitability of the materials to various applications, as an increase in the surface affects the dynamics of cell interactions.30 The findings of this study are useful in deciding on the correct choice of a bone substitute in view of the specific clinical situation and the resulting requirements. Here, particular attention has to be directed to the major criterion of material degradation versus material preservation. On the basis of our results, the application of Cerabone or NanoBone, which exhibited low ion release even in the early investigation period, would appear to be beneficial for the preparation of implant insertions due to their high volume stability, which has also been substantiated in vivo.31–33 On the other hand, Maxresorb revealed high ion disposal, which would indicate a correlation with its rapid resorption and osseous reorganization in vivo.34 In contrast to resistant bone substitutes, materials belonging to this subgroup would be of benefit in clinical situations where an orthodontic space closure upon previous bone grafting is desired.35 Although we chose early observation periods for our investigation due to the clinical limitations of the in vitro experimental setup, our results nevertheless reflect the effects of these two different types of bone substitute material. On the basis of the macro- and microstructure analyses of the bone substitutes, different material characteristics were uncovered, which will help improve our understanding of the fundamental processes involved in osseous tissue regeneration. Thus, our study may facilitate the development of standards in the choice of an appropriate bone substitute material with regard to patient-specific clinical needs, with the application of either a stable placeholder, or a bioactive degradable material.
Bone substitute resorption
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Figure 3. Material composition analysis of (a) Cerabone, (b) Maxresorb, and (c) NanoBone. Energy-dispersive X-ray spectroscopy (EDX) analyses confirmed Ca and P as the main structural substrates of the three bone substitutes evaluated. Maxresorb and Cerabone had obvious peaks for C, P, and O. NanoBone displayed a large peak for Si and a smaller one for C.
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Funding
None.
8.
Competing interests
Prof. Go¨tz and Dr. Reichert gave lectures for Artoss in the year 2012. All other authors disclose no potential conflicts of interest.
9.
10.
Ethical approval
Not required.
11.
Patient consent
Not required. 12.
Acknowledgement. The authors would like to thank J. Scheld (Institute of Geology and Mineralogy, University of Cologne, Cologne, Germany) for technical support. 13.
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Address: Christoph Reichert Department of Orthodontics University of Bonn
Welschnonnenstr. 17 D-53111 Bonn Germany Tel: +49 228 28722677; Fax: +49 228 28722455 E-mail: [email protected]
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Research Paper Bone Substitutes
Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells
A. Konermann1, M. Staubwasser2, C. Dirk3, L. Keilig3, C. Bourauel3, W. Go¨tz1, A. Ja¨ger1, C. Reichert1 1
Department of Orthodontics, Dental School, University of Bonn, Bonn, Germany; 2Institute of Geology and Mineralogy, University of Cologne, Cologne, Germany; 3Oral Technology, Dental School, University of Bonn, Bonn, Germany
A. Konermann, M. Staubwasser, C. Dirk, L. Keilig, C. Bourauel, W. Go¨tz, A. Ja¨ger, C. Reichert: Bone substitute material composition and morphology differentially modulate calcium and phosphate release through osteoclast-like cells. Int. J. Oral Maxillofac. Surg. 2014; 43: 514–521. # 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved. Abstract. The aim of this study was to determine the material composition and cellmediated remodelling of different calcium phosphate-based bone substitutes. Osteoclasts were cultivated on bone substitutes (Cerabone, Maxresorb, and NanoBone) for up to 5 days. Bafilomycin A1 addition served as the control. To determine cellular activity, the supernatant content of calcium and phosphate was measured by inductively coupled plasma optical emission spectrometry. Cells were visualized on the materials by scanning electron microscopy. Material composition and surface characteristics were assessed by energy-dispersive X-ray spectroscopy. Osteoclast-induced calcium and phosphate release was material-specific. Maxresorb exhibited the highest ion release to the medium (P = 0.034; calcium 40.25 mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (P = 0.021; calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (P = 0.034; calcium 16.34 mg/l day 5, phosphate 30.6 mg/l day 5). All investigated materials showed unique resorption behaviours. The presented methodology provides a new perspective on the investigation of bone substitute biodegradation, maintaining the material-specific micro- and macrostructure.
Bone is a dynamic tissue remodelled by osteoblast–osteoclast interactions under the regulation of the RANK/RANKL/ OPG system.1 In bony defect healing, bone grafting materials are the most important acquirement in osseous tissue regeneration. The osteoconductive and 0901-5027/040514 + 08 $36.00/0
osteoinductive features of these materials are controlled by different inherent material characteristics as much as by environmental criteria.2 The most important requirements for an ideal bone substitute are biocompatibility, osteoinduction, and osteoconduction, and for dental implants,
Key words: bone substitutes; biodegradation; calcium; osteoclastic cells; phosphate. Accepted for publication 17 October 2013 Available online 20 November 2013
stable long-term osseointegration with the re-establishment of structural tissue to attain a ‘restitutio ad integrum’.3 Previous in vitro research has mainly focussed on osteoblast–material interactions,4–6 and investigations of bone substitute biodegradation have principally
# 2013 International Association of Oral and Maxillofacial Surgeons. Published by Elsevier Ltd. All rights reserved.
Bone substitute resorption been restricted to animal experiments.7,8 Osteoclastic bone resorption occurs following strong attachment of the osteoclast to the hard tissue surface and subsequent generation of an acidic pH of 4.5 within the ‘ruffled border’, a special cellular compartment, to solubilize the alkaline salts of the bone mineral.9 Prior investigations of bone graft–osteoclast interactions have tended to study the cellular characteristics of bone remodelling processes,10–12 but the physico-chemical dissolution behaviour of bone substitutes has yet to be fully determined. Bone substitute materials include both synthetic and biological substitutes, and are characterized by their chemical composition, crystallinity, and morphology13; these have to meet specific criteria, as defined by their surface structure and configuration, in order to mimic in vivo effects.14 Calcium (Ca)–phosphate (P) bioceramics of synthetic or natural origin have unique compositions analogous to bone mineral and its specific turnover, which involves, among other processes, physiological Ca and P metabolism.14 However, the specific mechanisms that underlie these remodelling processes have yet to be fully elucidated. The aim of this study was to quantify the cell-mediated dissolution behaviour of three different bone substitutes in vitro by osteoclasts, with regard to Ca and P release from the bone substitutes. Furthermore, the cell adhesion qualities and the material composition of the bone substitutes were investigated. Materials and methods
Cell culture
A RAW 264.7 murine monocytic/macrophagic-type cell line (CLS Cell Line Services GmbH, Eppelheim, Germany) was seeded at a density of 3 107 cells/ml and cultured with 20 ng/ml macrophage colony-stimulating factor (M-CSF; BioCat GmbH, Heidelberg, Germany) and 30 ng/ml RANKL (Axxora GmbH, Lo¨rrach, Germany) in Dulbecco’s modified Eagle’s medium (DMEM; Invitrogen, Carlsbad, CA, USA) supplemented with 10% heat-inactivated foetal calf serum (FCS; Invitrogen) and 1 mg/ml penicillin/streptomycin (Invitrogen) at 37 8C in a humidified 5% CO2 atmosphere for osteoclast differentiation, as described previously.19,20 After 12 days, tartrateresistant acid phosphatase (TRAP) staining, as well as measurement of osteoclastic marker expression (TRAP, cathepsin K, vitronectin receptor) was performed to determine the osteoclastic phenotype (data not shown). The cells were then passaged into 24-well tissue culture plates (Greiner Bio-One International AG, Kremsmu¨nster, Austria), covered with or without different Ca–P-based bone substitutes in 2 ml medium/well. All materials were obtained directly from the manufacturers in sealed vials and were used without further modification. Control assays were performed with the addition of 100 nM bafilomycin A1 (Sigma–Aldrich, St. Louis, MO, USA) to wells containing osteoclasts, in order to mediate inhibition of active endocytosis by RAW cells (data not shown).21,22 Samples with medium alone served as reference controls.
Bone substitutes
Three different bone substitutes were included in this study, one of bovine origin and two synthetic composite materials: Cerabone1 (Botiss Dental GmbH, Berlin, Germany), a high-temperature sintered material of bovine origin enclosing highly crystalline hydroxyapatite (HA) as the sintered inorganic part of bovine bone, with 50% porosity and particle sizes of up to 1 mm15; Maxresorb1 (Botiss Dental GmbH, Berlin, Germany), a synthetic material composed of 60% HA/40% btricalcium phosphate (b-TCP) with macropores of 200–800 mm and micropores of 1–10 mm16; NanoBone1 (Artoss GmbH, Rostock, Germany), a bone substitute based on nanocrystalline HA embedded in a nanostructured silica, which has been described to undergo complete biodegradation by osteoclasts in vivo.7,8,17,18
Sample preparation
After 1 and 5 days, cell culture supernatants were harvested and transferred into glass vials for further treatment. Samples were dried using a red light (150 W) under a laboratory fume hood; 0.2 ml of aqua regia was then added (mixed from 3 parts 65% hydrochloric acid and 1 part nitric acid; Merck KGaA, Darmstadt, Germany) and the samples left for 24 h. This was followed by a final dilution in 3 ml Ampuwa water (Fresenius Kabi Germany GmbH, Bad Homburg, Germany) to filter out any residue with a particle diameter over 1.2 mm remaining after sample drying (FP 30 1.2CA; Schleicher & Schuell BioScience GmbH, Dassel, Germany) in glass containers (bottles with a rolled edge, 3 ml; VWR International GmbH, Darmstadt, Germany).
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Inductively coupled plasma optical emission spectrometry analyses
Ca and P ion concentrations in the samples (dilution 1:10) were measured with an inductively coupled plasma optical emission spectrometer (Ametek, Spectro A. I. Inc., Mahwah, NJ, USA) and an SC-4 DXS autosampler (Elemental Scientific, Omaha, NE, USA) at wavelengths of 317.933 nm (Ca) and 177.495 nm (P). The control program of the mass spectrometer performed three consecutive measurements of each sample, delivering the mean and standard deviation of the three results. Ion concentrations were calculated by inductively coupled plasma optical emission spectrometry (ICP-OS), which was calibrated by a standard control dilution series of defined ion concentrations subjected to matrix adjustment, delivering calibration curves as a reference. The detection limit and high resolution were guaranteed by continuous unit calibration before measurement of the first sample and after every 10 samples, and by repeated measurements of random samples as quality control. Scanning electron microscopy analyses
After removal of the supernatants for ICPOS analyses, the cells cultivated on bone substitutes for 5 days, as well as the surfaces of the three bone substitutes, were visualized with a scanning electron microscope (SEM, XL30; Philips, Eindhoven, the Netherlands) operating at 10 kV. For this purpose, the cells were fixed with paraformaldehyde (2.5%, pH 7) at 4 8C for 24 h and dehydrated in a graded alcohol series for 1 h each (30%, 50%, 70%, 80%, 90%, and 99.6%). The samples were then immersed in 1,1,1,3,3,3-hexamethyldisilizane (HDMS; Carl Roth GmbH, Karlsruhe, Germany) for 5 min, air dried at room temperature, and mounted on stainless steel stubs with double sticky tabs. The conduction was increased with Leit-C (Conductive Carbon Cement; Plano, Wetzlar, Germany) and sputter coating with platinum (Sputter Coater S150B; Edwards Ltd, Crawley, UK). Energy-dispersive X-ray spectroscopy analyses
To assess the material composition of the three different bone substitutes, energydispersive X-ray spectroscopy (EDX) analyses were performed using a GENESIS 4000 EDX system (EDAX, Mahwah,
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NJ, USA) attached to the scanning electron microscope described above. For the EDX scans, an acceleration voltage of 20 kV and a magnification of 1000 were used in the SEM. The scan was performed over the whole visible region of the sample, thus determining the average element distribution in the region studied.
Table 1. ICP-OS ion concentration measurements.a Ion Ca
Results ICP-OS analyses
The absolute values of the ICP-OS ion concentration measurements are listed in Table 1. Measurement of Ca and P ion concentrations in samples containing bone substitutes in medium alone showed a slight initial decrease in the basal ion concentrations induced by Cerabone, NanoBone, and Maxresorb after 1 day of incubation. Ca concentrations declined from 27 mg/l as the standard medium control value to 29% thereof for NanoBone, 64% for Maxresorb, and 75% for Cerabone. Similarly, P concentrations diminished from 43 mg/l as the pure medium control value to 23% thereof for NanoBone, 79% for Cerabone, and 88% for Maxresorb. At day 5, these levels remained relatively stable for both Ca and P. Taken together, Maxresorb showed the highest Ca and P medium content, followed by Cerabone and NanoBone. Analyses of the influence of osteoclasts on the dissolution behaviour of bone substitutes showed an initial Ca and P decrease in the medium on day 1, as seen for bone substitutes in medium alone. In combination with Cerabone, the presence of osteoclasts led to lower ion levels compared to the bone substitute alone, whereas incubation of osteoclasts with NanoBone resulted in higher Ca and P levels. For Maxresorb, ion concentrations were lower in the presence of cells on day 1, but had become higher on day 5 compared to the bone substitute incubated alone. In detail, Cerabone and NanoBone exhibited similar concentrations developing for both Ca and P and a subsequent
BS
BS + OC + bafilomycin
P
Concentration (mg/l)
Material
BS + OC
Statistical analysis
Statistical tests were performed using GraphPad Prism software version 4 (MacKiev Software). Data are expressed as the mean standard deviation. Statistical significance was assessed by Student’s t-test for unpaired observations after verifying normal distribution of the values. P < 0.05 was considered statistically significant.
Condition
BS BS + OC BS + OC + bafilomycin
Day 1
Day 5
Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone
20.12 17.28 7.71 18.13 18.90 7.81 16.42 15.03 9.08
16.60 17.03 6.46 16.34 40.25 8.43 13.63 29.46 12.06
Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone Cerabone Maxresorb NanoBone
34.11 37.98 9.82 29.26 39.78 11.02 26.74 26.10 11.12
31.10 55.18 12.77 30.60 102.08 15.15 26.09 66.42 17.21
ICP-OS, inductively coupled plasma optical emission spectrometry; BS, bone substitute; OC, osteoclasts. a Absolute values of the ICP-OS ion concentration measurements are presented in mg/l. Data represent the mean values of three independent measurements for each sample.
Table 2. P-values for the analysis of the ICP-OS measurements: comparison between Cerabone, NanoBone, and Maxresorb for the measured Ca and P ion concentrations on day 1 and day 5.
Cerabone Cerabone Maxresorb
P
Ca
Material Maxresorb NanoBone NanoBone
Day 1
Day 5
Day 1
Day 5
0.773 0.021* 0.021*
*
*
0.034* 0.021* 0.034*
0.034 0.021* 0.034*
0.021 0.021* 0.021*
ICP-OS, inductively coupled plasma optical emission spectrometry. * P < 0.05.
Table 3. P-values for the analysis of the ICP-OS measurements: comparison between day 1 and day 5 for the measured Ca and P ion concentrations with Cerabone, NanoBone, and Maxresorb. Material Cerabone Maxresorb NanoBone
Ca (day 1–day 5)
P (day 1–day 5)
0.248 0.034* 0.773
0.386 0.034* 0.149
ICP-OS, inductively coupled plasma optical emission spectrometry. * P < 0.05.
decline to the initial levels on day 5. Maxresorb manifested a different dissolution pattern, as Ca increased to 149% and P to 238% of the medium control value on day 5. Statistical analysis revealed significant differences in both Ca and P content in the supernatants between the materials investigated and at both observation timepoints, except Cerabone in comparison to Maxresorb for Ca on day 1. Furthermore, Maxresorb showed statistically
significant differences in the measured values of both ions when compared between day 1 and day 5. Calculated Pvalues are listed in Tables 2 and 3. Data on the outcomes when bafilomycin A1 was added, showed that Ca and P release were suppressed by the inhibition of cellular activity due to exposure to the substrate. Only cells incubated with NanoBone could not be blocked effectively by bafilomycin, which became particularly evident on day 5 (Fig. 1).
Bone substitute resorption
Figure 1. Ca and P levels induced by bone substitutes and by bone substitutes cultured with osteoclasts. Results of the inductively coupled plasma optical emission spectrometry (ICP-OS) measurements of the Ca (a) and P (b) ion concentrations in samples containing DMEM with Cerabone, NanoBone, or Maxresorb, with or without osteoclasts, after 5 days of incubation. Measurements were taken at wavelengths of 317.933 nm for Ca and 177.495 nm for P. Concentrations are expressed in mg/l and represent the mean values of three independent measurements.
SEM analyses
EDX analyses
SEM analyses highlighted the different ultrastructures of the three bone substitutes investigated. Cerabone presented a compact but ruffled surface, with round protrusions observed at the highest magnification. In contrast to NanoBone and Maxresorb, this material had only a few pores. In the overview, Maxresorb was found to be widely covered with pores and NanoBone was covered with pores of different sizes. The highest magnification revealed that both materials were covered with micropores over the whole surface. Cellular adhesion to the surface was evident for all the materials investigated, and the cells appeared to be vital (Fig. 2).
EDX analyses confirmed Ca and P as the main structural substrates of the three bone substitutes evaluated (Fig. 3). In addition, NanoBone was shown to incorporate appreciable amounts of carbon (C) and silicate (Si). The perceptual net weights of the different materials were: Cerabone C 9.44, N 2.71, O 29.97, Na 0.24, Si 0.61, P 18.25, Ca 38.73; Maxresorb C 7.95, N 4.98, O27.5, Na 0.71, Si 0.23, P 17.93, Ca 40.69; NanoBone C13.2, N 3.81, O 32.61, Na 0, Si 10.01, P 11.62, Ca 28.75. Discussion
Bone grafting materials are the most important acquirement in osseous tissue
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regeneration, with autogenous bone still representing the gold standard due to its osteogenic, osteoinductive, and osteoconductive qualities and high biocompatibility.15 As its availability is limited, bone substitutes with identical or at least analogous capacities are useful alternatives to autogenous bone.13,15 The aim of this study was to elucidate the biochemical properties of three different Ca–P-based bone substitutes with regard to material characteristics, Ca and P metabolism, and interaction with osteoclasts. Ca–P bioceramics are subject to two basic degradation mechanisms, namely hydrolysis of Ca and P by extracellular fluids and cell-mediated degradation by macrophages, polymorphonuclear giant cells, and osteoclasts.23 The degraded products are resorbed and transcytosed to the extracellular space,9,24 which enables the concentration changes in Ca and P in culture supernatants to be assessed as an indicator of osteoclastic activity. In our experiments, an initial decrease in Ca and P content in the supernatants both with and without osteoclasts was evident and remained relatively stable over time. Maxresorb exhibited the highest ion content in the medium (calcium 40.25 mg/l day 5, phosphate 102.08 mg/l day 5) and NanoBone the lowest (calcium 8.43 mg/l day 5, phosphate 15.15 mg/l day 5); Cerabone was intermediate (calcium 16.34 mg/l day 5, phosphate 30.60 mg/l day 5). In vivo, bone and bone substitute degradation is regulated by both cellular function and the interaction of a plurality of proteins, e.g. osteopontin, secreted by macrophages,25,26 whose cofactorial effects are difficult to replicate in an in vitro approach. However, our experimental setup maintained the specific features of the commercially available bone substitutes in accordance with their in vivo application. Other study designs have been based on morphological or morphometric analyses of osteoclasts or resorption pits on flat ceramic surfaces, thus discarding the specific structure of the bone substitute.27 All the materials investigated in this study show a specific micro- and macrostructure, supposedly exerting different effects on the dissolution behaviour of Ca and P. These ions, which have been proved to be a prerequisite for the process of osteoinduction, were determined to be the main structural substrates of the three bone substitutes evaluated, and NanoBone, in addition, contained considerable fractions of
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Figure 2. Ultrastructure and cell affinity of Cerabone, NanoBone, and Maxresorb. Representative visualization of RAW 264.7 cells on the bone substitute after 5 days. As well as the cellular morphology, the macrostructure of the different materials is evident. (a) Cerabone presented a compact, but ruffled surface. The cells were widespread and had thin protrusions. Several accumulations of cells located in resorption pits were visible. (b) In the ultrastructure, Maxresorb was covered with small pores. The cellular adhesion on Maxresorb was rather spare. Multinuclear cells were evident in large resorption pits. (c) NanoBone was ruffled and the material showed an inhomogeneous structure. It was completely penetrated by sponge-like holes. The cells on the material showed a strong polarization, and strong attachment to the surface was evident.
C and Si. Ca and P release by bone substitutes after implantation is followed by precipitation of an apatite layer due to supersaturation of these ions in the concavities of the material, presumably inducing osteoblastic cell formation directly or indirectly via the activation of growth factors.28,29 Our SEM analyses showed Cerabone to possess a ruffled surface with round protrusions and few pores, whereas Maxresorb was widely covered with pores and NanoBone was covered with pores of different sizes. These specific morphologies appear to determine the suitability of the materials to various applications, as an increase in the surface affects the dynamics of cell interactions.30 The findings of this study are useful in deciding on the correct choice of a bone substitute in view of the specific clinical situation and the resulting requirements. Here, particular attention has to be directed to the major criterion of material degradation versus material preservation. On the basis of our results, the application of Cerabone or NanoBone, which exhibited low ion release even in the early investigation period, would appear to be beneficial for the preparation of implant insertions due to their high volume stability, which has also been substantiated in vivo.31–33 On the other hand, Maxresorb revealed high ion disposal, which would indicate a correlation with its rapid resorption and osseous reorganization in vivo.34 In contrast to resistant bone substitutes, materials belonging to this subgroup would be of benefit in clinical situations where an orthodontic space closure upon previous bone grafting is desired.35 Although we chose early observation periods for our investigation due to the clinical limitations of the in vitro experimental setup, our results nevertheless reflect the effects of these two different types of bone substitute material. On the basis of the macro- and microstructure analyses of the bone substitutes, different material characteristics were uncovered, which will help improve our understanding of the fundamental processes involved in osseous tissue regeneration. Thus, our study may facilitate the development of standards in the choice of an appropriate bone substitute material with regard to patient-specific clinical needs, with the application of either a stable placeholder, or a bioactive degradable material.
Bone substitute resorption
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Figure 3. Material composition analysis of (a) Cerabone, (b) Maxresorb, and (c) NanoBone. Energy-dispersive X-ray spectroscopy (EDX) analyses confirmed Ca and P as the main structural substrates of the three bone substitutes evaluated. Maxresorb and Cerabone had obvious peaks for C, P, and O. NanoBone displayed a large peak for Si and a smaller one for C.
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Funding
None.
8.
Competing interests
Prof. Go¨tz and Dr. Reichert gave lectures for Artoss in the year 2012. All other authors disclose no potential conflicts of interest.
9.
10.
Ethical approval
Not required.
11.
Patient consent
Not required. 12.
Acknowledgement. The authors would like to thank J. Scheld (Institute of Geology and Mineralogy, University of Cologne, Cologne, Germany) for technical support. 13.
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Address: Christoph Reichert Department of Orthodontics University of Bonn
Welschnonnenstr. 17 D-53111 Bonn Germany Tel: +49 228 28722677; Fax: +49 228 28722455 E-mail: [email protected]
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